title: Truncated Type II isopentenyl diphosphate isomerase from hyperthermophilic Achaeon Thermococcus kodakaraensis implicates the necessity of its N-terminal amino acid residues in protein thermostability.
abstract: The enzyme isopentenyl diphosphate isomerase (IDI, EC 5.3.3.2) interconverts isopentenyl diphosphate and dimethylallyl diphosphate. We had previously cloned Tk-idi gene encoding the thermostable Tk-IDI enzyme from Thermococcus kodakaraensis KOD1. Four putative start codons were found on Tk-idi gene at 123, 213, 297 and 321 positions downstream of the first start codon. In the present work four mutants were obtained by deleting 123, 213, 297 and 321 nucleotides from the 5'-end of Tk-idi gene to obtain Tk-idim, Tk-idim1, Tk-idim2, and Tk-idim3, respectively. When we tried to express these truncated genes in Escherichia coli only Tk-idim was expressed in the active form. The product, Tk-IDIM, was purified and characterized. The molecular mass of the enzyme, estimated by gel filtration chromatography, was 300 kDa which indicated that the truncated enzyme retained the octameric form. The removal of 41 N-terminal amino acids did not exhibit a significant effect on the enzyme activity however, the thermostability of the enzyme decreased. The decrease in thermostability of Tk-IDIM correlated well with the results of circular dichroism (CD) analysis and structural modeling.
authors.affiliation: Department of Chemistry, Biotechnology Research Laboratory, University of Balochistan, Quetta, Pakistan. masoodbioeng@hotmail
authors.forename: Masood Ahmed
authors.initials: MA
authors.lastname: Siddiqui
authors.forename: Naeem
authors.initials: N
authors.lastname: Rashid
authors.forename: Habibur
authors.initials: H
authors.lastname: Rehman
mesh_terms: D000595:Amino Acid Sequence; D019748:Carbon-Carbon Double Bond Isomerases / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D045782:Hemiterpenes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055550:Protein Stability; D013329:Structure-Activity Relationship; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D045782:Hemiterpenes; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase